Posttranscriptionally modified nucleotides in RNA are generally conserved and phylogenetically distinct, although knowledge of distribution and sequence locations is restricted to relatively few organisms. Progress in recent years has begun to unfold the functional roles of modifications, including maintenance of reading frame during protein synthesis, stabilization of RNA secondary and tertiary structure and influence on RNA-protein interactions. We propose to apply techniques based on electrospray ionization mass spectrometry directly combined with liquid chromatography, recently developed in this laboratory to expand knowledge in selected areas concerning the identities and potential functions of RNA modifications. Of particular interest are tRNA modifications resulting from novel biosynthetic pathways, e.g., the action of aminoacyl tRNA synthetase paralogs and modifications which influence amino acid charging of tRNAs. [unreadable] [unreadable]